It is now believed that the cytoskeleton is widely used to transport mRNAs between their transcription and processing sites in the nucleus and their translation and degradation sites in the cytoplasm (Pachter, 1992; Bassell and Singer, 1997; Nakielny et al., 1997). One consequence of the interaction between mRNAs and the cytoskeleton is to promote differential localization and/or transport of mRNAs in subcellular compartments. Indeed, examples of mRNA targeting were observed in both germinal and somatic cells throughout the animal kingdom (Wilhelm and Vale, 1993; St Johnson, 1995; Steward, 1997). The universal use of this mechanism is also apparent when we consider the nature of the proteins which are coded by the transported mRNAs; asymmetric localization involving mRNAs coding for cytosolic, secreted, membrane-associated or cytoskeletal proteins have all been reported. Localization of mRNAs in the cytoplasm is now considered an essential step in the regulation of gene expression and an efficient way to unevenly distribute proteins in polarized cells. In general, it is believed that mRNA localization is used to determine and/or regulate local sites of translation (Rings et al., 1994; St Johnston, 1995; Steward, 1997). Indeed, ribosomes and many translational cofactors were found in association with the cytoskeletal elements, preventing both mRNAs and translation factors from being diluted by the cellular fluid (Pachter, 1992). Transport and local translation of specific mRNAs has been shown to play an important role in processes such as learning and memory (Martin et al., 1997), synaptic transmission (Crino and Eberwine, 1996; Kang and Schuman, 1996; Gazzaley et al., 1997; Steward, 1997; Tongiorgi et al., 1997), axis formation during development (reviewed in St Johnston, 1995), cell motility (Kislauskis et al., 1997), and asymetric cell division (Li et al., 1997; Long et al., 1997; Takizawa et al., 1997; Broadus et al., 1998).
The mechanisms underlying mRNA localization are not yet fully understood, mainly because of the lack of information on the principal constituents of the ribonucleoprotein complexes involved in this process. Nevertheless, it is known to involve both cis-acting signals in mRNA and trans-acting RNA-binding proteins which bind to this signal (St Johnston, 1995). The signals that allow mRNAs to be recognized as a target for transport and then to be localized have been mapped within their 3′-untranslated regions (Wilhelm and Vale, 1993; St Johnston, 1995). In contrast, the nature of the RNA-binding proteins is still obscure. Recently, a 68 kDa protein which binds the β-actin mRNA zipcode localization domain was isolated and its transcript was cloned from chicken cDNA libraries (Ross et al., 1997). This protein, which binds to microfilaments, contains an RNA-binding domain which shares strong sequence similarity with the RNP1 and RNP2 motifs. In addition, 69 kDa and 78 kDa proteins in Xenopus oocyte extracts have been shown to bind to the localization signal of Vg1 mRNA (Schwartz et al., 1992; Deshler et al. 1997). While the 69 kDa protein was shown to bind microtubules (Elisha et al., 1995), the 78 kDa Vera protein co-localized with a sub-domain of the smooth endoplasmic reticulum (Deshler et al., 1997). However, since these proteins have not yet been characterized, their nature and function in localization remain unclear.
Genetic and molecular studies have shown that the activity of the staufen gene product in Drosophila is necessary for the proper localization of bicoid and oskar mRNAs to the anterior and posterior cytoplasm of oocytes, respectively, and of prospero mRNA in neuroblasts (St Johnston et al., 1989; Ephrussi et al., 1991; Kim-Ha et al., 1991; St Johnston et al., 1991; Broadus et al., 1997; Li et al., 1997). Staufen is a member of the double-stranded RNA-binding protein family, and contains three copies of a domain consisting of a 65- to 68-amino acid consensus sequence which is required to bind RNAs having double-stranded secondary structures, and two copies of a short-domain, which retains the last 21 amino acids at the C-terminal end of the complete motif (St Johnston et al., 1991; St Johnston et al., 1992). In vitro, it has been demonstrated that staufen binds directly to bicoid and prospero mRNAs (St Johnston et al., 1992; Li et al., 1997). However, since staufen seems to bind to any dsRNA in vitro, it is not clear whether or not it binds directly to these RNAs in vivo, or needs cellular co-factors which make up part of a larger ribonucleoprotein complex to localize each mRNA. Many experiments have demonstrated that the localization of oskar, prospero and bicoid mRNAs occurs through a multistep mechanism of active transport that is dependent on elements of the cytoskeleton (Erdelyi et al., 1995; Pokrywka and Stephenson, 1995; St Johnston, 1995; Tetzlaff et al., 1996; Broadus et al., 1997).
There thus remains a need to understand the mechanisms of mRNA transport in mammals and determine the nature of both the RNAs and proteins in the RNA/protein complexes. Recently, both Southern blot analysis of human DNA and fluorescent in situ hybridization (FISH) on human chromosomes in metaphase showed that the human gene is present as a single copy in the human genome and is localized in the middle of the long arm of chromosome 20 (DesGroseillers and Lemieux, 1996). The identification and characterization of human (or another mammalian) staufen is desired as it could provide critical information in the transport, and proper localisation of mRNAs in subcellular compartments.
Staufen (Stau) was originally described as a dsRNA-binding protein in Drosophila melanogaster (1). It was further shown to specifically bind the 3′ untranslated region of the mRNA for bicoid (2), a morphogen responsible for anterior body pattern formation in the early embryo. In Drosophila, Stau's principle function is to target mRNAs for localized translation (2, 3): it serves to localize oskar mRNA posteriorally (3) and anchors bicoid mRNA anteriorally in oocytes, and recently has been shown to localize prospero mRNA in neuroblasts (4). The human homologue (hStau) is hereinbelow further characterized and is shown to have several structural and functional domain similarities to its Drosophila counterpart (5).
A more thorough understanding of the structure-function relationship of mammalian staufen is needed to better understand its function in mammalian cells. There also remains a need to better understand the dsRNA-binding activity of mammalian staufen and to analyze the function and application thereof in cellular homoeostasy. In addition, this understanding could help characterize the important molecular determinants of staufen from lower eukaryotes.
It would be highly desirable to be provided with means to target molecules to RNA viruses, including retroviruses, such as HIV virions. It would also be desirable to be provided wth means to target molecules into such viruses and affect their structural organization and/or functional integrity and/or morphogenesis.
It would also be highly desirable to be provided with a protein, fragment or derivative thereof which permits the development of chimeric molecules that can be specifically targeted into RNA viruses in general, and more particularly retroviruses, including antiviruses such as HIV. Such chimeric molecules could be used for the treatment of RNA virus infections, retroviral infections and lentiviral infections.
It would also be highly desirable to be provided with a therapeutic agent which permits targeting of chimeric molecules into RNA virions, as a treatment for diseases caused by such virions.
It would also be highly desirable to be provided with the identification of novel molecular determinants responsible for the incorporation of proteins into virions via their interaction with genomic RNA, for RNA genome incorporation into RNA viruses, as well as the identification of molecular determinants involved in the targeting of RNA molecules to the RER.
It would also be highly desirable to be provided with means to target RNA molecules to the RER.
It would also be very desirable to be provided with therapeutic agent molecules which interfere with the molecular determinant responsible for RNA genome incorporation into RNA virions as well as agents which interfere with the targeting of RNA molecules to the RER as such agents could have therapeutic utility for the treatment of diseases including viral diseases.
It would further be highly desirable to be provided with an assay which enables the screening and identification of molecules which modulate the interaction between the molecular determinant responsible for RNA genome incorporation into RNA virions. As well, it would be highly desirable to be provided with an assay which enables the screening and identification of molecules which modulate the targeting of RNA molecules to the RER.
It would in addition be highly desirable to be provided with a method for screening and identifying molecules which act as modulating agents of RNA genome incorporation into RNA virions and as well as a method for screening and identifying molecules which act as modulating agents for the targeting of RNA molecules to RER.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference.